CN116949025A - Phenylalanine ammonia lyase mutant derived from candida nodosa and application thereof - Google Patents

Phenylalanine ammonia lyase mutant derived from candida nodosa and application thereof Download PDF

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CN116949025A
CN116949025A CN202311000694.4A CN202311000694A CN116949025A CN 116949025 A CN116949025 A CN 116949025A CN 202311000694 A CN202311000694 A CN 202311000694A CN 116949025 A CN116949025 A CN 116949025A
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nppal
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phenylalanine ammonia
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杨兆勇
金媛媛
魏西羽
樊帅
邹森
张倩
吕旭东
冯翠月
吕瑞杰
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Abstract

The invention provides a candida nodosa-derived phenylalanine ammonia lyase mutant and application thereof, and belongs to the technical field of biological medicines. The invention provides a candida nodosa-derived phenylalanine ammonia-lyase mutant, which is subjected to at least one of the following mutations on the basis of NpPAL with an amino acid sequence shown as SEQ ID NO: 1: the 5-10 amino acids at the N-terminal are correspondingly mutated into the 5-10 amino acids at the N-terminal of ApPAL, the N2K mutation, the I3T mutation and the T4L mutation. Compared with NpPAL, the recombinant expression protein collected by escherichia coli recombinant expression is a mutant, the mutant has great improvement on the activity of enzyme catalysis of phenylalanine to cinnamic acid, and meanwhile, the yield of soluble expression is also greatly improved, so that a new thought is provided for preparing medicines for treating phenylketonuria.

Description

Phenylalanine ammonia lyase mutant derived from candida nodosa and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a candida nodosa-derived phenylalanine ammonia lyase mutant and application thereof.
Background
Phenylketonuria (PKU) is an autosomal recessive genetic disease that results in elevated levels of phenylalanine in patients, causing serious neurological damage and mental well-being due to loss of function of phenylalanine hydroxylase (PAH). Since the first attempt to treat PKU patients on a low phenylalanine diet, treatment of PKU has made many advances. While attempts have been made to develop PAH as an enzyme replacement therapy, the need for tetrahydrobiopterin cofactors and the long-term residence of this enzyme in the cytoplasm of hepatocytes make PAH a less likely therapeutic approach. The us Food and Drug Administration (FDA) approved pegylated recombinant phenylalanine ammonia lyase (PEGylated recombinatedphenylanineammonialyases) from anabaena variabilis (Palynziqs, biomain pharmaceuticals inc.) in 2018 to treat PKU. As an enzyme replacement therapy, phenylalanine Ammonia Lyase (PAL) is effective in converting L-phenylalanine to non-toxic trans-cinnamic acid and reducing elevated levels of L-phenylalanine in the blood of PKU patients. In addition, stable PAL can be used as a potential therapeutic enzyme for cancer treatment because it has significant cytotoxic effects on different cell types.
Up to now, crystal structures of PAL from houttuynia cordata, candida nodosa and some other sources have been reported. The crystallographic structure of PAL structures with homotetrameric activity most common in yeasts, cyanobacteria and plants demonstrates that electrophilic prosthetic 4-methyleneimidazol-5-one (MIO) is critical for catalytic activity. MIO is formed by spontaneous cyclization and dehydration of a highly conserved Ala-Ser-Gly (alanine-serine-glycine) motif that attacks substrates to promote ammonia elimination without the need for exogenous cofactors. In homotetramers, each of four identical active sites is formed by three different monomer subunits. Structurally, PAL monomers consist mainly of alpha-helices, with five alpha-helices in the central bundle. A long hairpin loop joins the last two helices, forming an inter-subunit interface at the core of the homotetramer.
In contrast to existing structures of eukaryotic PALs, two fragments, i.e. a long N-terminal extension and a shielding domain inserted near the C-terminal, are deleted in blue algae-derived PALs, such as anabaena polytricha-derived PAL (AvPAL) and candida nodosa-derived PAL (NpPAL). Studies have shown that long N-terminal extensions can affect the conformation of the active site cap loop, affect PAL stability and activity, and that the inserted shielding domain is associated with PAL thermal stability. The N-terminal motifs of AvPAL and NpPAL constitute the majority of the subdomains containing MIO prosthetic groups. However, the function of the N-terminal fragment of cyanobacteria PAL is still unknown. Although AvPAL and NpPAL have high sequence identity and tertiary structure, there are significant differences in their activity and soluble heterologous expression yield in e.coli. Given the great importance of AvPAL as a therapeutic in the treatment of phenylketonuria, there is a need to engineer NpPAL proteins in order to produce soluble and active recombinant proteins for use in the treatment of diseases.
Disclosure of Invention
In view of the above, the present invention aims to provide a candida nodosa-derived phenylalanine ammonia lyase mutant which has significantly improved enzyme activity and soluble expression compared with the original NpPAL.
The invention provides a candida nodosa-derived phenylalanine ammonia-lyase mutant, which is subjected to mutation of at least one of the following sites on the basis of NpPAL with an amino acid sequence shown as SEQ ID NO: 1: N2K mutation, I3T mutation, T4L mutation, L6Q mutation, Q7A mutation and N9S mutation.
Preferably, when three sites are mutated simultaneously, the phenylalanine ammonia lyase mutant is 5-Av-NpPAL;10-Av-NpPAL;
the amino acid sequence of the 5-Av-NpPAL is shown in SEQ ID NO. 3;
when 6 sites are mutated simultaneously, the phenylalanine ammonia lyase mutant is 10-Av-NpPAL;
the amino acid sequence of the 10-Av-NpPAL is shown as SEQ ID NO. 5.
Preferably, when only one site is mutated, the phenylalanine ammonia-lyase mutant comprises NpPAL-N2K, npPAL-I3T and NpPAL-T4L;
the amino acid sequence of the NpPAL-N2K is shown as SEQ ID NO. 7;
the amino acid sequence of the NpPAL-I3T is shown as SEQ ID NO. 9;
the amino acid sequence of NpPAL-T4L is shown as SEQ ID NO. 11.
The invention provides a gene for encoding the candida utilis-derived phenylalanine ammonia-lyase mutant.
Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO. 12.
The invention provides a recombinant expression vector, which comprises an expression vector of the gene.
The invention provides a recombinant engineering strain, which comprises the gene or the recombinant expression vector.
The invention provides a preparation method of a candida nodosa-derived phenylalanine ammonia-lyase mutant, which comprises the following steps:
the gene or the recombinant expression vector is introduced into a host strain, and the recombinant protein is isolated through culturing and induced expression.
The invention provides a medicament for treating phenylketonuria, which comprises a candida punctata-derived phenylalanine ammonia lyase mutant and auxiliary materials.
The invention provides an application of the candida nodosa-derived phenylalanine ammonia-lyase mutant, the recombinant expression vector, the recombinant engineering strain or the candida nodosa-derived phenylalanine ammonia-lyase mutant prepared by the preparation method in preparing medicaments for preventing and/or treating phenylketonuria.
The invention provides a candida nodosa-derived phenylalanine ammonia-lyase mutant, which is subjected to mutation of at least one of the following sites on the basis of NpPAL with an amino acid sequence shown as SEQ ID NO: 1: N2K mutation, I3T mutation, T4L mutation, L6Q mutation, Q7A mutation, and N9S mutation; the amino acid sequence of the ApPAL is shown as SEQ ID NO. 20. Experiments prove that after the mutation transformation, the mutant provided by the invention has greatly improved enzyme catalytic activity compared with NpPAL, and the enzyme activity is improved by 1.5-3 times; meanwhile, the solubility of the modified mutant is also greatly improved, and the mutant is expressed by adopting a prokaryotic expression system, so that the yield of the soluble protein is greatly improved compared with that of NpPAL, and the yield of the soluble protein is improved by 50-300%. Therefore, the mutant provided by the invention has the characteristics of high enzyme activity and strong solubility, and can be used for preparing a substitute medicine for treating phenylketonuria.
Drawings
FIG. 1 shows the results of the assay for the activity of NpPAL and its mutants;
FIG. 2 shows a Bradford method protein standard curve;
FIG. 3 shows the results of soluble protein production assays for NpPAL and its mutants.
Detailed Description
The invention provides a candida nodosa-derived phenylalanine ammonia-lyase mutant, which is subjected to mutation of at least one of the following sites on the basis of NpPAL with an amino acid sequence shown as SEQ ID NO. 1 (MNITSLQQNITRSWQIPFTNSSDSIVTVGDRNLTIDE VVNVARHGTQVRLTDNADVIRGVQASCDYINNAVETAQPIYGVTSGFGGMADVVISREQAAELQTNLIWFLKSGAGNKLSLADVRAAMLLRANSHLYGASGIRLELIQRIETFLNAGVTPHVYEFGSIGASGDLVPLSYITGALIGLDPSFTVDFDGKEMDAVTALSRLGLPKLQLQPKEGLAMMNGTSVMTGIAANCVYDAKVLLALTMGVHALAIQGLYGTNQSFHPFIHQCKPHPGQLWTADQMFSLLKDSSLVREELDGKHEYRGKDLIQDRYSLRCLAQFIGPIVDGVSEITKQIEVEMNSVTDNPLIDVENQVSYHGGNFLGQYVGVTMDRLRYYIGLLAKHIDVQIALLVSPEFSNGLPPSLVGNSDRKVNMGLKGLQISGNSIMPLLSFYGNSLADRFPTHAEQFNQNINSQGYISANLTRRSVDIFQNYMAIALMFGVQAVDLRTYKMKGHYDARTCLSPNTVQLYTAVCEVVGKPLTSVRPYIWNDNEQCLDEHIARISADIAGGGLIVQAVEHIFSSLKST): N2K mutation, I3T mutation, T4L mutation, L6Q mutation, Q7A mutation and N9S mutation. The amino acid sequence of ApPAL is shown in SEQ ID NO. 20 (MK TLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVARVARNGTLVSLTNNTDILQGIQASCDYINNAVESGEPIYGVTSGFGGMANVAISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMRGASGIRLELIKRMEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTGIAANCVYDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMISLLANSQLVRDELDGKHDYRDHELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYIGLLAKHLDVQIALLASPEFSNGLPPSLLGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIFQNYVAIALMFGVQAVDLRTYKKTGHYDARACLSPATERLYSAVRHVVGQKPTSDRPYIWNDNEQGLDEHIARISADIAAGGVIVQAVQDILPCLH).
In the present invention, the N-terminal 5-10 amino acid corresponding mutation is preferably an N-terminal 5-10 amino acid mutant of ApPAL, and comprises at least one of the following: 5-Av-NpPAL, 6-Av-NpPAL, 7-Av-NpPAL, 8-Av-NpPAL, 9-Av-NpPAL and 10-Av-NpPAL; the amino acid sequence of the 5-Av-NpPAL is shown as SEQ ID NO. 3 (MKTLSLQQNITRSWQIPFTNSSDSIVTVGD RNLTIDEVVNVARHGTQVRLTDNADVIRGVQASCDYINNAVETAQPIYGVTSGFGGMADVVISREQAAELQTNLIWFLKSGAGNKLSLADVRAAMLLRANSHLYGASGIRLELIQRIETFLNAGVTPHVYEFGSIGASGDLVPLSYITGALIGLDPSFTVDFDGKEMDAVTALSRLGLPKLQLQPKEGLAMMNGTSVMTGIAANCVYDAKVLLALTMGVHALAIQGLYGTNQSFHPFIHQCKPHPGQLWTADQMFSLLKDSSLVREELDGKHEYRGKDLIQDRYSLRCLAQFIGPIVDGVSEITKQIEVEMNSVTDNPLIDVENQVSYHGGNFLGQYVGVTMDRLRYYIGLLAKHIDVQIALLVSPEFSNGLPPSLVGNSDRKVNMGLKGLQISGNSIMPLLSFYGNSLADRFPTHAEQFNQNINSQGYISANLTRRSVDIFQNYMAIALMFGVQAVDLRTYKMKGHYDARTCLSPNTVQLYTAVCEVVGKPLTSVRPYIWNDNEQCLDEHIARISADIAGGGLIVQAVEHIFSSLKST); the amino acid sequence of the 10-Av-NpPAL is shown in SEQ ID NO. 5 (MKTLSQAQSKTRSWQIPFT NSSDSIVTVGDRNLTIDEVVNVARHGTQVRLTDNADVIRGVQASCDYINNAVETAQPIYGVTSGFGGMADVVISREQAAELQTNLIWFLKSGAGNKLSLADVRAAMLLRANSHLYGASGIRLELIQRIETFLNAGVTPHVYEFGSIGASGDLVPLSYITGALIGLDPSFTVDFDGKEMDAVTALSRLGLPKLQLQPKEGLAMMNGTSVMTGIAANCVYDAKVLLALTMGVHALAIQGLYGTNQSFHPFIHQCKPHPGQLWTADQMFSLLKDSSLVREELDGKHEYRGKDLIQDRYSLRCLAQFIGPIVDGVSEITKQIEVEMNSVTDNPLIDVENQVSYHGGNFLGQYVGVTMDRLRYYIGLLAKHIDVQIALLVSPEFSNGLPPSLVGNSDRKVNMGLKGLQISGNSIMPLLSFYGNSLADRFPTHAEQFNQNINSQGYISANLTRRSVDIFQNYMAIALMFGVQAVDLRTYKMKGHYDARTCLSPNTVQLYTAVCEVVGKPLTSVRPYIWNDNEQCLDEHIARISADIAGGGLIVQAVEHIFSSLKST).
In the present invention, the phenylalanine ammonia-lyase mutant preferably includes NpPAL-N2K, npPAL-I3T and NpPAL-T4L; the amino acid sequence of the NpPAL-N2K is shown as SEQ ID NO. 7 (MKITSLQQNITRSWQIPFTNSSDSIVTVGDRNLTIDEVVNVARHGTQVRL TDNADVIRGVQASCDYINNAVETAQPIYGVTSGFGGMADVVISREQAAELQTNLIWFLKSGAGNKLSLADVRAAMLLRANSHLYGASGIRLELIQRIETFLNAGVTPHVYEFGSIGASGDLVPLSYITGALIGLDPSFTVDFDGKEMDAVTALSRLGLPKLQLQPKEGLAMMNGTSVMTGIAANCVYDAKVLLALTMGVHALAIQGLYGTNQSFHPFIHQCKPHPGQLWTADQMFSLLKDSSLVREELDGKHEYRGKDLIQDRYSLRCLAQFIGPIVDGVSEITKQIEVEMNSVTDNPLIDVENQVSYHGGNFLGQYVGVTMDRLRYYIGLLAKHIDVQIALLVSPEFSNGLPPSLVGNSDRKVNMGLKGLQISGNSIMPLLSFYGNSLADRFPTHAEQFNQNINSQGYISANLTRRSVDIFQNYMAIALMFGVQAVDLRTYKMKGHYDARTCLSPNTVQLYTAVCEVVGKPLTSVRPYIWNDNEQCLDEHIARISADIAGGGLIVQAVEHIFSSLKST); the amino acid sequence of the NpPAL-I3T is shown as SEQ ID NO. 9 (MNTTSLQQNITRSWQIPFTNSSDSIVTVGDRNLTIDEVVNVARHGT QVRLTDNADVIRGVQASCDYINNAVETAQPIYGVTSGFGGMADVVISREQAAELQTNLIWFLKSGAGNKLSLADVRAAMLLRANSHLYGASGIRLELIQRIETFLNAGVTPHVYEFGSIGASGDLVPLSYITGALIGLDPSFTVDFDGKEMDAVTALSRLGLPKLQLQPKEGLAMMNGTSVMTGIAANCVYDAKVLLALTMGVHALAIQGLYGTNQSFHPFIHQCKPHPGQLWTADQMFSLLKDSSLVREELDGKHEYRGKDLIQDRYSLRCLAQFIGPIVDGVSEITKQIEVEMNSVTDNPLIDVENQVSYHGGNFLGQYVGVTMDRLRYYIGLLAKHIDVQIALLVSPEFSNGLPPSLVGNSDRKVNMGLKGLQISGNSIMPLLSFYGNSLADRFPTHAEQFNQNINSQGYISANLTRRSVDIFQNYMAIALMFGVQAVDLRTYKMKGHYDARTCLSPNTVQLYTAVCEVVGKPLTSVRPYIWNDNEQCLDEHIARISADIAGGGLIVQAVEHIFSSLKST); the amino acid sequence of the NpPAL-T4L is shown as SEQ ID NO. 11 (MNILSLQQNITRSWQIPFTNSSDSIVTVGDRNLTIDEV VNVARHGTQVRLTDNADVIRGVQASCDYINNAVETAQPIYGVTSGFGGMADVVISREQAAELQTNLIWFLKSGAGNKLSLADVRAAMLLRANSHLYGASGIRLELIQRIETFLNAGVTPHVYEFGSIGASGDLVPLSYITGALIGLDPSFT VDFDGKEMDAVTALSRLGLPKLQLQPKEGLAMMNGTSVMTGIAANCVYDAKVLLALTMGVHALAIQGLYGTNQSFHPFIHQCKPHPGQLWTADQMFSLLKDSSLVREELDGKHEYRGKDLIQDRYSLRCLAQFIGPIVDGVSEITKQIEVEMNSVTDNPLIDVENQVSYHGGNFLGQYVGVTMDRLRYYIGLLAKHIDVQIALLVSPEFSNGLPPSLVGNSDRKVNMGLKGLQISGNSIMPLLSFYGNSLADRFPTHAEQFNQNINSQGYISANLTRRSVDIFQNYMAIALMFGVQAVDLRTYKMKGHYDARTCLSPNTVQLYTAVCEVVGKPLTSVRPYIWNDNEQCLDEHIARISADIAGGGLIVQAVEHIFSSLKST).
The invention provides a gene for encoding the candida utilis-derived phenylalanine ammonia-lyase mutant.
In the present invention, the nucleotide sequence of the gene is preferably shown as SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO. 12.
The invention provides a recombinant expression vector, which comprises an expression vector of the gene.
The present invention is not particularly limited in the kind of expression vector, and expression vectors well known in the art may be used, including prokaryotic expression vectors. In the embodiment of the invention, pET21a (+) is adopted as the expression vector. Cloning sites are preferably Nde I and Xho I. The construction method is not particularly limited, and construction methods known in the art may be employed, including cleavage and ligation, and verification.
The invention provides a recombinant engineering strain, which comprises the gene or the recombinant expression vector.
In the present invention, the host strain of the recombinant engineering strain preferably comprises a prokaryotic expression system. In the examples of the present invention, E.coli (Escherichia coli OverExpress C (DE 3)) was used as the host strain. The preparation method of the recombinant engineering strain is not particularly limited, and transformation methods well known in the art can be adopted.
The invention provides a preparation method of a candida nodosa-derived phenylalanine ammonia-lyase mutant, which comprises the following steps:
the gene or the recombinant expression vector is introduced into a host strain, and the recombinant protein is isolated through culturing and induced expression.
In the invention, the gene for encoding the phenylalanine ammonia lyase mutant is preferably obtained by performing point mutation by using an amplification primer on the basis of taking the encoding gene of the phenylalanine ammonia lyase as a template;
the forward primer for amplifying the gene encoding 5-Av-NpPAL is a primer with a nucleotide sequence shown in SEQ ID NO:14, 5-Av-NpPAL-F;
the forward primer for amplifying the gene encoding 10-Av-NpPAL is a primer with a nucleotide sequence shown in SEQ ID NO: 15-Av-NpPAL-F;
the forward primer for amplifying the gene encoding NpPAL-N2K is a nucleotide sequence shown in SEQ ID NO:16, npPAL-N2K-F;
the forward primer for amplifying the gene encoding NpPAL-I3T is a nucleotide sequence shown in SEQ ID NO:17, npPAL-I3T-F;
the forward primer for amplifying the gene encoding NpPAL-T4L is a nucleotide sequence shown in SEQ ID NO:18, npPAL-T4L-F;
the universal reverse primer is a nucleotide sequence shown in SEQ ID NO:19, npPAL-R.
The forward primer for amplifying the gene encoding the NpPAL is a primer with a nucleotide sequence shown in SEQ ID NO:13, npPAL-F.
In the present invention, the method of introduction is preferably a heat shock method. The culture conditions are preferably 200 r.min -1 Culturing at 37deg.C to OD 600 When the value is 0.4-0.6, the expression is induced. The culture is performed by adopting an LB culture medium containing Amp. The induced expression is preferably added to a final concentration of 0.2 mmol.L -1 isopropyl-beta-D-thiogalactoside (IPTG) and shaking culture at 16℃for a further 16h.
In the invention, the activities of the mutants 5Av-NpPAL, 10Av-NpPAL, N2K, T I and T4L prepared by the method are respectively improved by 2 times, 2.9 times, 2 times, 1.7 times and 2.2 times compared with the activities of the NpPAL, and the modified mutants are greatly improved in the aspect of catalyzing the activity of phenylalanine to cinnamic acid. At the same time, the yield of soluble proteins is also substantially improved, and in particular, the soluble protein expression yields of mutants 5Av-NpPAL, 10Av-NpPAL, N2K and T4L are respectively improved by 92%, 190%, 50% and 69% compared with the NpPAL.
The invention provides a medicament for treating phenylketonuria, which comprises a candida punctata-derived phenylalanine ammonia lyase mutant and auxiliary materials.
The invention has no special limitation on the types of the auxiliary materials, and the auxiliary materials of protein medicines known in the art can be adopted. The preparation method of the medicine is not particularly limited, and the preparation method of protein medicines known in the art can be adopted.
In view of the good solubility expression capability and the good enzyme catalytic activity of the candida nodosa-derived phenylalanine ammonia-lyase mutant, the invention provides the candida nodosa-derived phenylalanine ammonia-lyase mutant, the recombinant expression vector, the recombinant engineering strain or the candida nodosa-derived phenylalanine ammonia-lyase mutant prepared by the preparation method and the application of the candida nodosa-derived phenylalanine ammonia-lyase mutant in preparing medicaments for preventing and/or treating phenylketonuria.
The phenylalanine ammonia lyase mutant derived from candida nodosa and the application thereof provided by the invention are described in detail below with reference to examples, but are not to be construed as limiting the scope of the invention.
Example 1
(1) Amplification of protein NpPAL mutant sequences
Site-directed mutagenesis was designed based on the amino acid sequence of NpPAL (SEQ ID NO: 1) and the nucleotide sequence of the coding sequence (SEQ ID NO: 2), and 5 mutants were designed, which were respectively 5Av-NpPAL (the amino acid sequence is shown as SEQ ID NO:3, the nucleotide sequence of the coding sequence is shown as SEQ ID NO: 4), 10Av-NpPAL (the amino acid sequence is shown as SEQ ID NO:5, the nucleotide sequence is shown as SEQ ID NO: 6), npPAL-N2K (the amino acid sequence is shown as SEQ ID NO:7, the nucleotide sequence is shown as SEQ ID NO: 8), npPAL-I3T (the amino acid sequence is shown as SEQ ID NO:9, the nucleotide sequence is shown as SEQ ID NO: 10) and NpPAL-T4L (the amino acid sequence is shown as SEQ ID NO:11, and the nucleotide sequence is shown as SEQ ID NO: 12). The PCR amplification is performed by designing an upstream primer and a downstream primer for site-directed mutation amplification. The specific primer sequences are as follows:
NpPAL-F：GAAGGAGATATACATATGAACATTACCAGCCTGCAGC
(SEQ ID NO:13)；
5Av-NpPAL-F：GAAGGAGATATACATATGAAAACCCTGAGCCTGCAG CAGAAC(SEQ ID NO:14)；
10Av-NpPAL-F：GAAGGAGATATACATATGAAAACCCTGAGCCAGGC GCAGAGCAAAACCCGCAGCTGG(SEQ ID NO:15)；
NpPAL-N2K-F：GAAGGAGATATACATATGAAAATTACCAGCCTGCAG CAG(SEQ ID NO:16)；
NpPAL-I3T-F：GAAGGAGATATACATATGAACACTACCAGCCTGCAG CAG(SEQ ID NO:17)；
NpPAL-T4L-F：GAAGGAGATATACATATGAACATTCTCAGCCTGCAG CAG(SEQ ID NO:18)；
NpPAL-R: GTGGTGGTGGTGCTCGAGGGTGCTTTTCAGGCTGCTA (SEQ ID NO: 19), wherein NpPAL-R is a universal downstream primer and the above-mentioned upstream primer are respectively subjected to PCR amplification.
The PCR amplification system is as follows: KOD FX Neo 1. Mu.L, 2X PCR Buffer for KOD FX Neo. Mu.L, ddH 2 O17. Mu.L dNTPs 4. Mu.L (concentration: 2 mM), template DNA 1. Mu.L (about 50 ng-200 ng), and each of the upstream and downstream primers 1. Mu.L (concentration: 20. Mu.M). The PCR reaction conditions were: pre-denaturation at 94 ℃ for 2min; denaturation at 94℃for 15s, annealing at 58℃for 15s, elongation at 68℃for 30s,30 cycles; finally, extending at 68 ℃ for 7min; preserving at 4 ℃.
(2) Construction of recombinant plasmid of NpPAL mutant
After the PCR amplification, the target band was rapidly cut out under an ultraviolet lamp at 120V for 40min by 0.7% agarose gel electrophoresis detection, a target DNA fragment of about 1700bp was recovered according to the specification of a gel recovery kit (Beijing Kao is a century biotechnology Co., ltd.), double digestion was performed with Nde I and Xho I, the obtained DNA fragment was connected with vector pET21a (+) (An Nuolun biotechnology Co., ltd.), and double digestion verification was performed with restriction enzymes Nde I and Xho I. The digested products were subjected to agarose gel electrophoresis at a voltage of 120V, and the sizes of the fragments were confirmed to be about 1700bp and about 5400bp, respectively. The ligation products were then transformed into E.coli TOP10 competent cells (Shanghai Weidi Biotechnology Co., ltd.) and screened on LB plates containing ampicillin antibiotics.
And 5 single colonies are respectively picked on a plate after transformation for verification, meanwhile, the picked colonies are transferred to an ampicillin resistance plate culture medium for culture, PCR amplification is carried out by adopting the amplification primers, after the PCR amplification is finished, result detection is carried out by agarose gel electrophoresis, the strains with the strips are sent to a sequencing company for sequencing, and after the sequencing result is received, the sequencing result is compared with a target fragment sequence to observe whether the sequencing is correct or not.
(3) Obtaining of monoclonal cell lines of NpPAL and mutants thereof
Extracting plasmid from bacterial liquid with correct sequence by using plasmid extraction kit (Beijing kang is century biotechnology Co., ltd.), and transforming into expression host E.coli over express C43 (DE 3) according to conventional method
And (Novagen company) to obtain the protein NpPAL and the mutant escherichia coli engineering bacteria thereof.
(4) Purification and enrichment of NpPAL and mutants thereof
Single colony is selected and inoculated into a culture medium, and is cultured at 200 r.min < -1 > and 37 ℃ overnight, activated bacterial liquid is transferred into a 50mL fungus shaking tube for expansion culture, and then inoculated into 1L of strain containing Amp (100 mu g.mL) according to the ratio of 1:100 -1 ) In LB liquid medium of (C), culturing at 37 deg.C until OD 600 When the value is 0.4-0.6, the final concentration is 0.2 mmol.L -1 isopropyl-beta-D-thiogalactoside (IPTG), regulating shaking table to set 16 ℃, continuing shaking culture for 16h, centrifuging the culture at 4 ℃ and 6000 r.min < -1 > for 5min, and crushing the collected thalli by a high-pressure homogenizer. Centrifuging the crushed thallus at 4deg.C for 30min, filtering the supernatant with 0.45 μm filter membrane, and purifying with Co 2+ And (5) purifying by a column to obtain the target protein.
The AvPAL-containing fractions were pooled and concentrated to 500 to 1000. Mu.L using a 30kDa ultrafiltration concentration tube, and 10mL of protein buffer (20 mmol.L) -1 Tris-HCl，150mmol·L -1 NaCl, pH 8.0), continue 4000 r.min -1 Centrifugation was repeated 2 to 3 times until the protein buffer was completely replaced.
Example 2
NpPAL and mutant enzyme activity determination thereof
The experimental method comprises the following steps: the enzyme-catalyzed reaction was 450. Mu.L, and 5. Mu.g of protein was added to a reaction system containing 10 mmol.L -1 Phenylalanine, 50 mmol.L -1 The reaction was carried out in Tris-HCl (pH 7.5) at 40℃for 5min, and then 50. Mu.L of trichloroacetic acid (TCA) was added to terminate the reaction, 12000 r.min -1 Centrifuging for 5min, measuring absorbance at 290nm under an ultraviolet spectrophotometer, determining the amount of cinnamic acid generated by a standard curve, and calculating the PAL enzyme activity.
The results are shown in FIG. 1.
The activity of NpPAL and its mutants was determined, wherein the activity of mutants 5Av-NpPAL, 10Av-NpPAL, N2K, T3I and T4L was increased by 2-fold, 2.9-fold, 2-fold, 1.7-fold and 2.2-fold, respectively, compared to NpPAL.
Example 3
NpPAL and mutant soluble protein expression yield determination thereof
The experimental method comprises the following steps: protein concentration was measured by the Bradford method, 5 Xstandard solution was diluted to 1X, filtered and used for BSA bovine serum albumin with ddH 2 O is diluted into 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 mg.mL respectively -1 And respectively taking 300 mu L of the filtered solution and 3 mu L of diluted standard solution, adding the filtered solution and the diluted standard solution into a 96-well plate, uniformly mixing, measuring absorbance values at the wavelengths of 595nm and 450nm, and drawing a standard curve to calculate the target protein concentration.
The results are shown in Table 1 and FIG. 2.
TABLE 1Bradford method for determining protein concentration Standard Curve
The soluble protein expression yields of NpPAL and mutants thereof were determined, wherein the soluble protein expression yields of mutants 5Av-NpPAL, 10Av-NpPAL, N2K, T L and I3T were increased by 92%, 190%, 50%, 69% and-30%, respectively, compared to the AvPAL.
Example 4
Enzymatic kinetic assay of NpPAL and mutants thereof
The experimental method comprises the following steps: to evaluate the kinetic parameters Km and kcat of the enzyme, to dissolve in 50 mmol.L -1 10-20000. Mu. Mol.L of Tris-HCl (pH 7.5) -1 Phenylalanine as substrate was reacted with 5. Mu.g of protein, absorbance was measured, and K was calculated by Michaelis-Menten nonlinear fitting mapping method in GraphPadprism8.0 m And Vmax, and calculating the corresponding K according to formula I by measuring the protein concentration cat Values.
k cat ＝V max /[E]Total formula I
Analysis of results: the results of the enzyme kinetics are shown in Table 2.
TABLE 2 kinetic parameters of NpPAL and mutants thereof
The 5Av-NpPAL and 10Av-NpPAL mutants compare to the wild-type NpPALk cat Increases the catalytic constant (k) of the L-phenylalanine by 2.5 times and 3.5 times respectively cat /K m ) The increase is 70% and 1.6 times respectively. Simultaneous mutant NpPAL-N2K, npPAL-I3T and NpPAL-T4L k cat The increase is 2.1 times, 1.8 times and 2.3 times respectively. Although most NpPAL mutations exhibit varying degrees of K m An increase, indicating a decrease in the affinity of the mutant for L-phenylalanine, but due to k cat The increase is greater, resulting in an increase in catalytic performance.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A candida nodosa-derived phenylalanine ammonia-lyase mutant is characterized in that the mutant is subjected to at least one of the following sites on the basis of NpPAL with an amino acid sequence shown as SEQ ID NO. 1: N2K mutation, I3T mutation, T4L mutation, L6Q mutation, Q7A mutation and N9S mutation.
2. The candida punctata-derived phenylalanine ammonia-lyase mutant according to claim 1 wherein when there are three simultaneous mutations at three sites the phenylalanine ammonia-lyase mutant is 5-Av-NpPAL;10-Av-NpPAL;
the amino acid sequence of the 5-Av-NpPAL is shown in SEQ ID NO. 3;
when 6 sites are mutated simultaneously, the phenylalanine ammonia lyase mutant is 10-Av-NpPAL;
the amino acid sequence of the 10-Av-NpPAL is shown as SEQ ID NO. 5.
3. The candida punctata-derived phenylalanine ammonia-lyase mutant according to claim 1 characterized in that when only one site is mutated, the phenylalanine ammonia-lyase mutant comprises NpPAL-N2K, npPAL-I3T and NpPAL-T4L;
the amino acid sequence of the NpPAL-N2K is shown as SEQ ID NO. 7;
the amino acid sequence of the NpPAL-I3T is shown as SEQ ID NO. 9;
the amino acid sequence of NpPAL-T4L is shown as SEQ ID NO. 11.
4. A gene encoding the candida utilis-derived phenylalanine ammonia-lyase mutant according to any one of claims 1 to 3.
5. The gene according to claim 4, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO. 12.
6. A recombinant expression vector comprising the gene of claim 4 or 5.
7. A recombinant engineering strain comprising the gene of claim 4 or 5 or the recombinant expression vector of claim 6.
8. A method for preparing the candida punctata-derived phenylalanine ammonia-lyase mutant according to any one of claims 1 to 3, which is characterized by comprising the following steps:
introducing the gene of claim 4 or 5 or the recombinant expression vector of claim 6 into a host strain, culturing and inducing expression, and isolating the recombinant protein.
9. A medicament for treating phenylketonuria, which comprises a candida punctata-derived phenylalanine ammonia lyase mutant according to any one of claims 1-3 and an adjuvant.
10. Use of a candida nodosa-derived phenylalanine ammonia-lyase mutant according to any one of claims 1-3, a recombinant expression vector according to claim 6, a recombinant engineering strain according to claim 7 or a candida nodosa-derived phenylalanine ammonia-lyase mutant prepared by the preparation method according to claim 8 in the preparation of a medicament for preventing and/or treating phenylketonuria.
CN202311000694.4A 2023-08-10 2023-08-10 Phenylalanine ammonia lyase mutant derived from candida nodosa and application thereof Pending CN116949025A (en)

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